Over the last decade, surface plasmon resonance (SPR) microscopy has emerged as a broadly applicable analytical tool for detecting and characterizing chemical and physical changes occurring in a highly localized probed region. Compatible with a wide range of condensed phase and gas phase environments, SPR is a virtually universally applicable sensing platform. In addition, SPR methods provide high detection sensitivities and excellent temporal resolution, and are compatible with a number of high throughput screening systems, such as microfluidic, nanofluidic and microarray systems. As a result of these beneficial attributes, a great deal of attention has been directed over the last several decade at developing SPR-based sensors capable of real time characterization of the composition, physical properties and optical properties (e.g., index of refraction) of a range of materials in a variety of media.
Surface plasmons (SPs), also know as surface plasmon waves and plasmon polaritons, are charge density waves, which propagate parallel to an interface between a conducting or semiconducting thin film and a dielectric layer. Surface plasmon resonance (SPR) microscopy uses resonant excitation of SPs to detect and characterize chemical and physical changes occurring in a probed region proximate to the sensing surface of thin metal or semiconductor films. In these systems, SPs are generated by coupling radiant energy from incident photons into the oscillating modes of free electrons present in a conducting material. Conventional SPR sensors achieve photon to SP coupling by interaction of evanescent electromagnetic waves generated via total internal reflection with a thin metallic or semiconducting layer. SPs are localized at the surface of the conducting (including semiconducting) layer and the intensity of the electric field of a SP decays exponentially in directions perpendicular to the plane in which it propagates. The highly localized nature of SP's excited by evanescent electromagnetic waves makes this technique ideally suited for sensing changes in a spatially localized probe region proximate to (<about 500 nanometer) the SPR sensing surface.
In recent years, SP sensors have been used extensively to characterize chemical and physical properties of a variety of biological materials. For example, surface plasmon resonance (SPR) instruments are widely used to study macromolecular biological interactions. Important applications of this technology include proteomics, virology, cell signaling, DNA hybridization, antibody-antigen interactions, DNA-protein interactions, and drug discovery. Commonly, these SPR applications involve sensing specific and/or nonspecific protein interactions with surface immobilized target species by detecting them on the basis of the sensitivity of SPR to even minute changes in refractive index occurring near the surface of a conducting material. Accordingly, SPR provides a virtually universally applicable form of chemical sensing, that can be effected without the need for prior spectroscopic (e.g., fluorescent) labeling. SPs have also been used, however, as a means of locally exciting fluorescent materials, such as fluorescently labeled probes. In these systems, energy from SPs is coupled to electron transitions in a fluorescent species in a manner providing highly localized excitation and emission.
Sensors based on SPR utilize the dependence of the SPR resonance condition on changes in the refractive index of a lower refractive index dielectric sample layer positioned adjacent to the thin electrically conducting film that supports SP generation. In typical sensing applications, changes in the resonance condition for formation of SPs are monitored in real time and directly related to chemical or physical changes occurring at an interface between the thin metal (or semiconductor) film and the media undergoing analysis. Sensors based on SPR may provide selective detection of materials and compounds by manipulating the chemical or physical properties of the sensing surface. In some applications, for example, the sensing surface of the SPR sensor is coated with a material exhibiting selective binding characteristics such that the refractive index varies in the presence of a specific material to be sensed. For example, the sensing surface may be made sensitive to a particular antibody by coating it with an antigen to that antibody. Using these principles, SPR detection has been successfully incorporated into a number of commercially available biological sensing devices including the sensors and screening devices manufactured by BIAcore, Inc.
Most commercial SPR systems use the Kretschmann configuration where a prism couples light, under total internal reflection, into surface plasmons (SPs) on flat, continuous gold films. In the Kretschmann geometry, evanescent electromagnetic waves penetrate the thin (≈50 nm) gold film(s) positioned between higher and lower refractive index dielectric layers and excite SPs, which propagate parallel to the outer surface of the metal film adjacent to a lower refractive index layer. The prism is needed in this configuration to achieve a wavevector matching condition between the incident polarized excitation light and the surface plasmons. For a given dielectric sample, photons of a certain wavelength and incident at a certain angle generate evanescent waves that penetrate the metal layer and excite surface plasmons at the metal-dielectric sample interface. Upon exciting SPs, the intensity of reflected light is reduced and, therefore, monitoring changes in the intensity of the reflected beam or measurement of the wavelength corresponding to the minimum intensity of reflected light can be used to detect change in refractive index in the localized probe region.
The Kretschmann SPR optical configuration, while highly sensitive (a Figure of Merit, FOM, of greater than about 1000 nm/RI is typical), is subject to several practical limitations. First, it is difficult to integrate theses optical configurations into complex form factor devices useful for high throughput fluidic screening applications, including microfluidic (μFl) and nanofluidic (nFl) systems for bioanalytical measurement. Kretschmann geometry based sensors assemblies using angle of incidence modulation require complex and spatially restrictive rotation assembles that are prohibitive for some applications and require frequent realignment and optimization. Second, the mechanical integrity of these systems may not be robust enough for important large-area, array-based screening applications. To address such limitations and extend the sensitivity of conventional SPR systems, specifically tailored nanoscale materials structures capable of generating plasmonic responses have attracted considerable attention for implementation in the next generation of SPR sensors.
Nanoparticle arrays, for example, can provide extremely sensitive and tunable plasmonic responses. These architectures require, however, external optical systems through which the array/nanoparticles can be spectroscopically interrogated, and therefore are less attractive for large-area, array-based screening applications. Further, significant deviations in nanoparticle positions and sizes provide practical limitations on the use of nanoparticle arrays for SPR sensing.
An alternative method of coupling incident electromagnetic radiation into plasmon modes involves the use of periodically structured metal thin films, such as nanohole arrays, where the coupling is dictated by the matching of photon and grating momenta, with the latter acting as its own optical device. Such plasmonic crystal structures, for example structures exhibiting enhanced transmission of light through their subwavelength apertures, appear promising for application in a range of optical technology areas, including sensing involving plasmonic responses. The nature and specific mechanisms of plasmon generation in these structures are subjects of substantial interest in research and, while incompletely understood, are known to involve the excitation of surface plasmon polaritons (SPPs) with contributions arising from surface plasmon resonances localized at the edges of the holes (LSPRs). The transmission spectra of these devices depend strongly on the properties of the fabricated holes (shape, size, period, structure) as well as the thickness of the metal and the index of the underlying and external media. While surface plasmon responses accessed using metal thin films is well established, application of these structures for SPR sensing has not yet been fully explored.
It will be appreciated from the foregoing that a clear need exists for methods and systems for generating plasmonic responses in thin electrically conducting films for use in sensing applications. SPR sensors are needed that are compatible with direct integration into complex device architectures such as microfluidic Lab on Chip (LOC) systems. SPR sensors are needed having form factors, sensitivities and time resolutions that are suitable for high throughput screening applications including implementation in fluidic and microarray systems. In addition, low cost, high precision and commercially practicable methods are required for fabricating SPR sensors, particularly SPR sensors based on plasmonic crystals.